(1) Summary of the Invention
The present invention relates to aerobic thermophiles which have the ability to use the monoterpenes, .alpha.- and .beta.-pinene, limonene, carveol, and carvone as sole carbon sources. The present invention also relates to the production of limonene, .beta.-pinene, .alpha.-terpineol, carvone, and carveol from monoterpene substrates using the aerobic thermophile isolate BR425. The present invention further relates to an isolated pinene hydroxylase that is a general allylic oxidizer.
(2) Description of Related Art
Pinenes, the major constituents of turpentine, are bicyclic monoterpenes which are produced in significant quantities by plants of the Pinaceae family. Because of their volitility, pinene emissions from conifer forests and during pulping operations constitute a major source of biogenic hydrocarbons (Lindskag and Potter, Chemosphere (1992), Vol. 30, pp. 1171-181; Stromvall and Petersson, Environ. Pollution (1993), Vol. 79, pp.219-223). Pinenes are useful for manufacture of a wide variety of consumer products. .alpha.-pinene is used to manufacture pine oil which is used in disinfectants, detergents mining chemicals, insecticides and resins. .beta.-pinene is a valuable raw material used in the manufacture of cosmetic fragrances and culinary flavorings. Pinenes are the major constituents of turpentine oils and consequently are very cheap to obtain and are readily available. Pinenes can be converted to several higher value monoterpenes such as carvone, .alpha.-terpineol and limonene which is usually performed by a series of complex synthetic chemical reactions (Kirk-Othmer, Encyclopedia of Chemical Technology, 3.sup.rd Edition., Vol. 22. (1983), Mark et al. Eds. John Wiley & Sons. New York, N.Y. pp. 709-749). Limonene is also a substrate for synthetic chemical production of monoterpenes such as carvone, perillyl alcohol and .alpha.-terpineol. It has been recognized that producing higher value monoterpenes from pinenes could be simplified if these compounds could be produced by microbial means.
U.S. Pat. No. 4,495,284 to Rhodes and Winskill describes the preparation of 1-carvone (spearmint flavor) by cultivating Pseudomonas strain NCIB 11671 in an aqueous nutrient media in the presence of 1-.alpha.- or 1-.beta.-pinene. However, Pseudomonas is not thermophilic and therefore cannot be cultivated under conditions favoring monoterpene volatilization which can be advantageous in bioprocessing applications. Furthermore, the strain was not disclosed as being capable of degrading other monoterpene substrates.
U.S. Pat. No. 5,487,988 to Chang and Oriel describes a process for the preparation of monoterpene compounds such as perillyl alcohol, perillyl aldehyde, and .alpha.-terpineol from limonene using thermophilic Bacillus stearothermophilus strain BR388. The Bacillus was not disclosed as being capable of degrading other monoterpene substrates such as pinenes.
While the above-mentioned U.S. Patent to Rhodes and Winskill discloses a strain of Pseudomonas capable of degrading pinenes and disclose use of the microorganism for producing 1-carvone from pinenes, the metabolism of pinenes by microorganisms has been little studied, since pinenes are both poorly soluble in aqueous media and membrane destructive, and therefore toxic to procaryotic and eucaryotic microorganisms (Andrews et al., Appl. Environ. Microbiol.(1980), Vol. 40, pp. 301-304).
In early reports, catabolism of .alpha.-pinene by Pseudomonas strain PL was suggested to proceed either by isomerization of the pinene to limonene with subsequent oxidation to perillic acid (Shukla and Bhattacharyya, Ind. J. Biochem.(1968), Vol. 5, pp 92-99) or by direct oxidation to pinene epoxide prior to ring cleavage (reviewed by Tudgill, Biodegradation, (1990), Vol. 1, pp. 93-1005). Recent evidence indicates the presence of a second silent pathway in one of the pinene-epoxidizing Pseudomonas strains, the metabolites for which have not been identified (Colocousi et al., Appl. Microbiol. Biotechnol. (1996), Vol. 45, pp. 822-830). To date none of the genes involved in the pinene catabolic pathway have been examined.
Thermophilic microorganisms have not been described that are capable of degrading pinenes at elevated temperatures which would allow production of higher value monoterpenes under conditions favoring monoterpene volatilization.